In order to understand how the nervous system functions it is necessary to understand the principles governing the development, maintenance and modification of synapses. A key element among synaptically-localized proteins is the neuronal nicotinic acetylcholine receptor (nAChR), the ion channel responsible for the transduction of signals between neurons. Although complementary DNA clones encoding nAChR subunits have been isolated and subsequent anatomical studies suggest differences in their cell-specific patterns of expression, the cellular and molecular mechanisms controlling the expression if these genes are unknown. Therefore, as part of a long term goal to understand the molecular events controlling nAChR expression, the specific aims of this proposal focus on identifying the molecular mechanisms controlling the expression of the gene encoding the beta4 subunit of the nAChR. Electrophysiological studies have shown that substituting beta4 for other beta subunits in the nAChR creates functionally distinct nAChR channels, while in situ hybridization suggests that the beta4 subunit gene exhibits a very restricted pattern of expression in the nervous system. Taken together, these two lines of evidence support the idea that functional heterogeneity of receptors in specific cell types is generated, at least in part, by transcriptional control of the nAChR subunit genes and suggest that the beta4 subunit gene is a particularly appropriate focus for initial attempts to understand the transcriptional control of the nAChR gene family. Therefore, genetic elements required for tissue-specific expression of the beta4 subunit gene will be identified and characterized, taking advantage of a 2.3 Kb genomic fragment of the 5' flanking region of the gene which we have recently isolated and have shown in preliminary experiments to confer cell- specific expression to a reporter gene in transfected mammalian cells. The deletional analysis of this fragment and identification of cis- acting elements necessary for its restricted expression will provide the first insight into the factors controlling the expression of the nAChR genes. Gel mobility shift, DNAse footprinting and methylation interference assays will be used to identify and characterize cellular factors that interact with these sequence elements and regulate beta4 expression in neuronal and non-neuronal cells. Finally, the mechanisms underlying the activation of the beta4 gene during neuronal differentiation will be investigated in PC12 cells, which, upon treatment with nerve growth factor, exhibit an increase in nAChR expression and an apparent increase in transcriptional activity of the beta4 gene. The results of these studies will provide valuable information regarding not only nAChR gene expression in the central nervous system, but also general principles of genetic regulation of neurotransmitter receptor diversity. This information will advance our understanding of the processes governing neuronal differentiation and development of the nervous system.